Abstract: The ovary is a crucial component of the female
reproductive system, playing key roles in follicular maturation
and production of hormones such as estradiol. AMPK is an enzyme
involved in protection of cells from metabolic stress, particularly
in liver and skeletal muscle. Preliminary studies in our laboratory
have shown mRNA for AMP-Activated Protein Kinase (AMPK) is expressed
in the rat ovary in a cell-specific manner, and suggest roles
of AMPK in regulating hormone production and cell viability.
The purpose of this study is to examine the presence of AMPK
±1 and ±2 subunit protein in the ovary. For immunoblot
analysis, whole ovary and liver were obtained from rats and proteins
extracted. Proteins were run through SDS-gel electrophoresis
and blotted onto a nitrocellulose membrane. Blots were incubated
with AMPK immunopurified goat anti-human AMPK ±1- and
±2-specific antibodies, and detected with rabbit anti-goat
secondary antibodies. Specific immunoreactive signal of the expected
molecular weights (60 kD) were observed in both ovary and liver
homogenates. To examine the cell-specific location of AMPK ±1
and ±2 subunit proteins in the ovary, immunohistochemical
studies are being performed. Twenty-one day old mice were treated
with eCG and hCG to induce follicle development and ovulation,
respectively, and ovaries collected at different time points
and fixed with paraformaldehyde. Ovaries were then processed
for immunohistochemical analysis, and 10 ºM paraffin sections
were places onto slides. Ongoing immunohistochemical analysis
with a confocal microscopy system will reveal the cell-specific
localization of AMPK ±1 and ±2 subunit proteins
in the ovary, providing new insight into the possible roles of
this enzyme in regulating reproductive functions.

Preparation and Characterization of a Silica Gel
Supported Zirconium(IV) Complex

Marisa Monreal

Abstract: The preparation and characterization of a
heterogeneous chiral Lewis acid catalyst, silica gel supported
diethyl-L-tartrate zirconium(IV), is reported. Early results
from [4+2] cycloaddition reactions of cyclopentadiene with methyl
acrylate catalyzed by II at room temperature demonstrate endo/exo
ratios similar to controls. Previous
work on the analogous homogeneous catalyst m-tolyl-tetraphenylcyclopentadienyl-zirconium
indicated a selectivity dependence on nature of dienophile and
temperature; these variables, in addition to new reactivity methods,
are being manipulated in the current system to maximize selectivity.
Elemental analysis, IR, and
solid-state NMR methods were used to confirm the structure of
II. These preliminary results are guiding our current work on
improving the synthesis and maximizing the selectivity of II,
as well as our investigations into derivatives of II.

Expression and Regulation of Adenylyl Cyclase Types
V and VI in the Rat Ovary

Anahid Mirzatoni, Dr. Philip S. LaPolt

Abstract:Adenylyl cyclases (ACs) are enzymes that convert
ATP into the second messenger cAMP. So far, nine isoforms of
ACs have been cloned in mammals and their expression has been
investigated in various tissues and cells. The regulation and
distribution of AC isoforms is very diverse and complex. It has
been shown that usually more than one isoform is expressed in
a tissue or cell. In ovarian granulosa cells, AC activity is
stimulated by follicle-stimulating hormone (FSH), resulting in
increased cAMP levels and production of the steroid hormone estradiol
(E2). Little is known regarding the regulated expression and
localization of AC isoforms in the ovary. This study tested the
hypothesis that AC isoforms V and/or VI are expressed in the
rat ovary in a cell-specific, regulated manner. RT-PCR confirmed
the presence of transcripts encoding AC V and VI isoforms in
whole ovary RNA. The identity of these PCR products was confirmed
using restriction mapping and DNA sequencing, and expression
was also confirmed by Northern blot hybridization analysis. Immunohistochemical
analyses of ACV/VI protein during ovarian development, ovulation,
and luteinization revealed localized expression of ACV/VI in
granulosa cells & oocytes of developing follicles, as well
as cumulus cells of preovulatory follicles. After ovulation,
ACV/VI was also detected in cells of the corpus luteum. These
findings provide new insights into the regulated expression of
ACV and VI in the rat ovary, and suggest that ACV/VI may play
roles in ovarian functions such as steroidogenesis, oocyte maturation
and function of the corpus luteum

Abstract: Jojoba oil is a wax ester that consists of
eicosenoic acid and eicosenol. Previous studies in our laboratory
have shown that female New Zealand White rabbits fed a 2% jojoba
oil diet for seven days have a significantly increased HDL-C
concentration. Our objective is to determine the optimal dietary
concentration of jojoba oil that increases HDL-C concentration
and the optimal time for maximizing the amount of eicosenoate
in the blood. Female New Zealand White rabbits were fed a rabbit
chow supplemented with 3 or 9% (w/w) jojoba oil for 39 days.
Blood was collected from an ear vein at 0, 14, 28, and 39 days.
Serum was obtained by centrifugation. Lipids were extracted from
serum, feces, and feed by the method of Bligh and Dyer, followed
by trans-esterification with sulfuric acid in methanol and quantification
of the fatty acid methyl esters with gas-liquid chromatography.
The HDL fraction was separated from the VLDL+LDL fraction by
polyanion precipitation of the serum, and total cholesterol concentration
was measured enzymatically. The highest concentration of HDL-C
in the blood occurs with the 3% jojoba oil diet, and the highest
concentration of eicosenoate in the blood occurs at 28 days.
Therefore, the 3% jojoba oil diet is better than the 9% diet
in altering HDL-C metabolism, and the optimal time for feeding
the diet is 28 days. (Supported by MBRS-RISE grant R25 GM61331.)

A New Tool to Study Processing of Human Defensin
5, An Important Mediator of Mucosal Immunity

Erika Reynoso

Abstract:A New Tool to Study Processing of Human Defensin
5, An Important Mediator of Mucosal Immunity
Paneth cells (PC) in the small intestine contribute to innate
mucosal immunity by secreting defensins, natural peptide antibiotics
that are small cationic amphipathic molecules with a membrane
disrupting activity. Many defensins are produced as prepropeptides
that undergo posttranslational sequential processing. Human defensin
5 (HD5) is found in PC granules as a propeptide (proHD5) and
is further processed by PC derived trypsin. As biological activities
of the different HD5 forms vary and processing may be altered
in diseases of the gastrointestinal tract, it is important to
have a tool to identify the various HD5 forms. We describe here
epitope mapping of monoclonal antibodies raised against proHD5
employing fluorimetric ELISA, dot blot and Western immunoblot
analysis. As antigens, recombinant and natural HD5 forms with
varying N-terminus and synthetic peptide fragments were used.
Polyclonal rabbit antisera raised against fully processed rHD5
reacted with all HD5 forms, except the synthetic fragments. Most
monoclonal forms were reactive only with incompletely processed
HD5 forms and non-reactive with fully processed HD5. These antibodies
will be useful in the study of HD5 processing and intestinal
diseases involving PC.

Abstract:Adenosine monophosphate-activated protein
kinase (AMPK) is a signaling enzyme activated by increased AMP
levels in liver and skeletal muscle, protecting cells against
metabolic stress associated with low ATP levels. AMPK is a heterotrimeric
protein kinase with multiple isoforms for each subunit (alpha,
beta, and gamma). Whereas only the alpha-1 and alpha-2 subunits
are catalytic, beta and gamma subunits are regulatory and necessary
for full enzymatic activity. Previous studies indicate that AMPK
is expressed in ovarian oocytes, and may play a role in oocyte
maturation. Preliminary data in our laboratory suggests a role
of AMPK in hormone production and viability of granulosa cells.
To better understand the role of AMPK in the ovary, we studied
the expression and potential function of AMPK alpha subunits
in cultured rat granulosa cells. Using reverse transcription-polymerase
chain reaction (RT-PCR) and specific primers for AMPK alpha-1
and alpha-2 subunits, PCR products corresponding to the expected
sizes were obtained, indicating the presence of AMPK alpha-1
and alpha-2 subunits in rat granulosa cells. To determine possible
functions of AMPK in granulosa cells, we are utilizing an antisense
oligonucleotide approach to inhibit alpha-1 and alpha-2 expression
in cultured cells. Culture of granulosa cells with a fluorescently
labeled version of this antisense oligonucleotide DNA revealed
uptake into cells, as determined by confocal microscopy. The
effectiveness of the antisense oligonucleotide in inhibiting
AMPK expression, and possible effects on hormone production,
are being determined. These studies demonstrate the expression
of AMPK alpha-1 and alpha-2 subunit mRNAs in rat granulosa cells,
and will provide new information regarding possible functions
of this enzyme in the ovary.

Abstract:The aggregation of _-synuclein, an amyloidogenic
protein, has been implicated as a critical step in the development
of Parkinson's disease (PD). Recently, it has been demonstrated
that amyloid proteins aggregate via partially folded intermediates
to form ordered aggregates, such as protofibrils and fibrils
as well as disordered amorphous aggregates. These _-synuclein
fibrils are found in the form of deposits in the cell. The molecular
mechanisms leading to protein deposition of _-synuclein are not
well understood. We hypothesize that interactions between _-synuclein
and certain surfaces may catalyze the aggregation process, such
as hydrophobic versus hydrophilic surfaces and positive versus
negative surfaces. We also believe that interactions between
_-synuclein and other molecules can inhibit or promote aggregation.
We plan to study these biomolecular interactions at the surface/solution
interface using atomic force microscopy (AFM), which is a powerful
technique for studying the morphology and structure of amyloid
fibrils, with high resolution and imaging ability in air and
fluid conditions. In addition, AFM is extremely powerful for
studying the amyloidogenic protein aggregation and fibrillation
at surfaces under physiologically relevant conditions. We would
like to obtain useful information about the factors leading to
_-synuclein protein aggregation so that mechanisms involving
the pathogenesis of Parkinson's disease can be formulated.

Abstract:Phosphorus is one of the most abundant nutrients
in the environment. However, even though it is abundant, little
is known about how it exists in nature. Past methods have not
specifically measured the different forms of inorganic phosphorus
(phosphate, phosphite, and hypophosphite) in natural waters.
Using ion chromatography, a new method has been developed that
is selective and can measure low concentrations of reduced phosphorus.
Water samples were collected from Hot Creek near Mammoth Lake,
CA during the summer of 2004. Preliminary analyses suggest these
samples may contain phosphite. The implication of this study
will be discussed.

Abstract:Siderophores are iron binding ligands, which
serve to gather iron for microbial cells. Under an iron deficient
environment bacteria produce and excrete siderophores. After
sequestering environmental ferric ion, these siderophores are
recognized and transported into the cell by receptors located
in the outer membrane. Escherichia coli (E.coli)
expresses on its surface the FepA receptor, which discriminately
transports iron through the siderophore enterobactin(1).
Studies have found that FepA also transports analogs of enterobactin
containing catecholate type binding units attached to simpler
backbones. Much is known about the fate and role of siderophores,
however little is known about the mechanistic interaction of
a siderophore during its transportation of iron. In order to
elucidate some of this we have proposed the use of bifunctional
analogs.
More specifically the utilization of adamantane derivative consisting
of three 2,3-dihydroxybenzoyl- arms in the 3,5,7-positions for
iron binding and a fourth position occupied by group R, which
potentially could be conjugated to fluorescent probe or an antibiotic
through a linker. Proton is used as a protecting group for unsymmetrical
amidation of tetra(aminomethyl)adamantane. Here we present synthesis
of tricatecholateadamantane(4) protected intermediate
starting from inexpensive adamantane.

Synthesis and Characterization of Pentaphenylcyclopentadienyltris(dimethylamido)
Zirconium and Chloride Derivatives

Rayshonda Williams

Abstract: We have prepared pentaphenylcyclopentadienyltris
(dimethylamido) zirconium (C5Ph5)Zr(NMe2)3 (I) and two
chloride derivatives: (C5Ph5)Zr(NMe2)2Cl (II) and (C5Ph5)Zr(NMe2)Cl2
(III) . I was synthesized by reacting pentapheylcyclopentadiene
(HC5Ph5) with tetrakisdimethylamidozirconium Zr(NMe2)4. Next,
II was produced by reacting I with one equivalent
of Me2NHoHCl which was added using a slow addition method at
room temperature. III was prepared by reacting I
with two equivalents of Me2NHo HCl which was added using the
same slow addition method but at -78oC. The compounds were characterized
by 1H and 13C NMR spectroscopy. They will be used in the synthesis
of chiral Lewis acids, which will be catalysts in C-C bond formation
reactions.

The Effect of Dietary Jojoba Oil on HDL Metabolism
in New Zealand White Rabbits

Maritza Hernandez, Monica Hernandez & Raymond
E. Garcia

Abstract: We have prepared pentaphenylcyclopentadienyltris
(dimethylamido) zirconium (C5Ph5)Zr(NMe2)3 (I) and two
chloride derivatives: (C5Ph5)Zr(NMe2)2Cl (II) and (C5Ph5)Zr(NMe2)Cl2
(III) . I was synthesized by reacting pentapheylcyclopentadiene
(HC5Ph5) with tetrakisdimethylamidozirconium Zr(NMe2)4. Next,
II was produced by reacting I with one equivalent
of Me2NHoHCl which was added using a slow addition method at
room temperature. III was prepared by reacting I
with two equivalents of Me2NHo HCl which was added using the
same slow addition method but at -78oC. The compounds were characterized
by 1H and 13C NMR spectroscopy. They will be used in the synthesis
of chiral Lewis acids, which will be catalysts in C-C bond formation
reactions.

Abstract:MT and FT immobilized onto a modified gold
surface were used as biosensors to detect metal ions such as
Cd, Zn by surface plasmon resonance. Injecting solutions of Cd
and Zn onto MT or FT we find that there is a conformational change
of the metalloproteins upon metal sequestration. Using 0.01 M
HCl for acidic conditions, we found that MT and FT can release
any metals bound to their cysteine groups, thereby, regenerating
the biosensor surface. Therefore, MT and FT as biosensor chips
have proved to be effective sensors for trace metals by SPR.

Preliminary Steps For The Recombination And Expression
of
Sulfiredoxin

Grace Aldana Masangkay

Abstract: Reactive oxygen species (ROS) such as hydrogen
peroxide (H2O2) can produce hydroxyl radicals (OHo) that damage
DNA. Oxidative damage affects the function or reproduction of
cells leading to diseases such as cancer and neurodegenerative
disorders. Living organisms have developed defense mechanisms
such as antioxidants that can prevent or repair oxidation. Peroxiredoxins
(prx), ubiquitous proteins that are found in organisms ranging
from bacteria to humans, can act as antioxidants reducing H2O2
to H2O. In eukaryotic cells, prx are also regulators of H2O2-mediating
signaling. In the presence of high levels of H2O2, peroxiredoxin
becomes overoxidized, forming sulfinic acid. The overoxidized
prx can no longer reduce H2O2 to H2O, allowing H2O2 to participate
in signaling. The formation of sulfinic acid was believed to
be irreversible; however, recent studies have shown that a yeast
protein called sulfiredoxin (srx) can reduce the cysteine-sulfinic
acid of yeast peroxiredoxin TsaI back to its thiol form, (Biteau,
B., Labarre, J., Toledano, M.B. (2003) Nature425,
980-984). . Our goal is to express srx cDNA (from cancerous human
lung tissue) in prokaryotic cells so that other lab members can
show that it can repair the oxidative damage in human peroxiredoxin
I. We plan to make a donor vector and fuse it with an acceptor
vector via Cre recombinase to form a recombinant plasmid that
will express the srx. Our project began by finding the sulfiredoxin
sequence on the NCBI website (AAH47707) and obtaining the srx
cDNA as an expressed sequence tagged DNA from Invitrogen. This
plasmid was transformed into E. coli cells and colonies were
picked and analyzed by restriction digestion. The srx coding
sequence was subcloned by Polymerase Chain Reaction (PCR) technique,
analyzed by agarose gel, and ligated into a plasmid to form a
donor vector. This vector was purified by column chromatography
and sequenced at City of Hope. In the future, we plan to form
a recombinant plasmid that will express the srx and use it to
repair overoxidized prx.

Synthesis and Characterization of a Photoreversible
Calcium Chelator Based on Azobenzene

Ruth Avila

Abstract:Calcium plays an integral role in cell signaling.
The release and uptake of calcium in an oscillatory manner triggers
various important physiological processes. In order to study
the effects of calcium oscillations in proteins and cells, calcium
signals will be mimicked artificially using a light-responsive
chelator. The aim of this project is to synthesize a molecule
based on azotoluene that can bind and release calcium reversibly
in response to irradiation. It is expected that different wavelengths
of light will isomerize the chelator, interconverting it between
high affinity (cis) and low affinity (trans) forms. This interconversion
should mimic biochemical calcium signals. The synthesis of this
molecule requires three steps. The first step is the synthesis
of 4,4'-dimethylazobenzene, which yielded 84.56% of expected
product. The second step is the synthesis of 4,4'-Bis(bromomethyl)
azobenzene, resulting in 73.40% yield of product, followed by
the third step: substitution of iminodiacetic acid for bromine,
which yielded 90.85% of pure product. Irradiation of the chelator
both in the presence and absence of metal ions such as calcium
resulted in the successful interconversion between cis and trans
forms. These findings form the basis for further studies involving
calcium binding and photochemistry in aqueous media, which will
be presented.

Poster
#15

The Effect of Low-Pass Filtering, Exposure, and
Delay on Voice Quality and Recognition

Shamaine Bonds

Abstract: Accuracy of voice recognition and perceptual
voice quality were tested for twelve digitally recorded female
voices. The task was to recognize whether the voices were
previously heard or not. Overall performance was 74% correct,
and significant effects were evident in voice quality ratings,
but not for delay, filtering, and exposure repetition.